JPH01137993A - Method for production of monoclonal antigram (-)antibody and method for its use - Google Patents

Abstract

PURPOSE: To obtain a monoclonal antibody of a mammal directed to a lipid A-core part in an endogenous toxin of various Gram-negative bacteria by using specific complete lipopolysaccharides as an antigen acting on immunization of an immunological competitive cell.

CONSTITUTION: Complete lipopolysaccharides of a Gram-negative bacterium in which a lipid A-core part is extracted are used as an antigen acting on the immunization of an immunological competitive cell. The complete lipopolysaccharides are preferably obtained from bacteria of the strains belonging to the genus Echerichia, Klebsiella, Pseudomonas, Enterobacter, Bacteroides, Serratia or Proteus.

COPYRIGHT: (C)1989,JPO

Description

DETAILED DESCRIPTION OF THE INVENTION [Technical Field to which the Invention Pertains] The present invention relates to a method for producing a monoclonal anti-Gram (-) antibody and its use.

[Prior art and its problems] Research conducted in the United States shows that gram (-)
Bacterial sepsis is constantly increasing.

A clinical trial conducted by Ziegler in 1982
We have demonstrated therapeutic improvements brought about by serum therapy in the treatment of Gram(-) sepsis and endotoxic shock.

Monoclonal antibodies (AcM) could play a role in serum therapy in the treatment of these diseases.

The endotoxin of Gram(-) bacteria is the antigenic lipopolysaccharide (L
PS).

Antibodies directed against the D antigen portion (ie, the D-side chain) of PS are specific for both the fungus and type of bacteria.

On the other hand, antibodies directed against the lipid A-core region of endotoxins in bacteria are likely to react with different types of Gram(-) bacteria. In fact, the lipid A-core moiety is relatively constant in Gram(-) bacteria.

Several publications describe monoclonal antibodies that can react with different types of Gram (-) bacteria.

PCT patent applications WO34104458 and WO351
No. 01659 and European Patent Application No. 174,20
No. 4 can be cited.

PCT application WQ 84/04458 uses endotoxins of [rough J mutants] as antigens, and these mutants lose antigen O and possibly the core sugar moiety to improve this specificity. is missing.

It is further stated that a partially degraded complete endotoxin can be used to obtain a simplified structure of the antigen in this type of endotoxin in a "crude" mutant form.

In fact, LPS has a hydrophobic moiety on the A lipid side and an O
It has a hydrophilic part on the antigen side. In aqueous media, these LPSs coalesce in a micelle structure, with the central lipid A moiety located in the center of the micelle and the antigen directed to the outside of the micelle.

Thus, the antigenic parts common to different species are hidden and unexposed and cannot generate non-specific antibodies. For this reason, an incomplete LPS is required.

This same approach has been demonstrated in PCT application WO 35/2013 using endotoxins of the LPS type from "crude" mutants as antigens.
It is also adopted in No. 01659.

This PCT application specifically describes a mixture of different monoclonal antibodies from mice that were protected against endogenous toxic shock caused by Escherichia coli J5 (ECJ5).

Contrary to accepted technical ideas, the applicant has obtained AcM that reacts with different types of Gram(-) bacteria;
As antigen a complete endotoxin from a smooth J bacterium was used, thus containing the antigen 0 portion.

[Problems to be Solved by the Invention] Therefore, an object of the present invention is to obtain a monoclonal anti-gram(-) antibody based on a concept different from that of the prior art. Furthermore, an object of the present invention is to obtain a method for producing this type of antibody and its use.

[Means for solving the problem] Therefore, the subject matter of the present invention is to solve the problems in different types of grams (
-) a method for producing mammalian heptclonal antibodies directed against the lipid A-core part of bacterial endotoxins;
□Antigen that acts on immunization of immune competing cells is lipid A-17
It is characterized by being a complete lipopolysaccharide of bacteria.

The term complete LPS refers to an endotoxin that includes all or most of the lipid A-core portion, the core portion, and antigen O.

When we say that the lipid A-core portion of LPS is exposed, this means that the lipid A-core portion of LPS, which is not generally exposed to contact with immune competing cells, is exposed and therefore has the ability to stimulate white blood cells B. means.

LPS can be obtained from any species of Gram(-) bacteria, preferably Klebsiella 1. It is obtained from the species of Escherichia, Pseudomonas, Enterobacter, Batatheroid, Serratia or Proteus, especially Klebsiella species, especially Klebsiella nilamonia, and Escherichia coli.

In preferred conditions for carrying out the above method, LPS has the structure L
It has PS-LAP. LAP stands for protein bound to lipids.Morrison: The Journal of
Immunology, Volume 119, No. 5 (November 1977)
(Mon.), p. 1700 and Handbook of Endotoxin, Vol. 1, "Chemistry of Endotoxin", Liechel Elsebir Publishing (11984), p. 339 et seq.: Protein Components of Bacterial Endotoxins].

LPS-LAP can be made according to methods described in the literature. Suitable LPS-LAPs are described in European Patent Nos. 49182 and 11598 for their preparation.
No. 8, these are called glycoproteins.

In particular, these involve culturing the microorganisms to full development, lysing the preferred microorganisms enzymatically and then chemically, drying the lysate, extracting the lipids with a solvent (preferably acetone and then methanol), and lysing the lysate. is suspended in water, physically deproteinized, preferably by centrifugation, and the solution is ultrafiltered under pressure through an ultrafilter, preferably with a separation range of 300,000, by quaternary ammonium halide. precipitate, preferably 5000 to 10
Concentrate the supernatant liquid using an ultrafilter with a separation range of 0.000 and determine LPS using a low molecular weight alcohol.
- Can be produced by precipitating LAP.

In vivo and in vitro methods for producing monoclonal antibodies are known in the literature, in particular in the above-mentioned PCT application W.
It is known from publications 034/04458 and 851016595.

Unlike the teachings of the prior art, immunization is carried out with complete LPS, but with its lipid A-core portion exposed. For example, mice are given one or more injections of LPS-LAP by the parenteral route (subcutaneously, intramuscularly or intraperitoneally), preferably at a body weight of 1 kg, in the presence or absence of an adjuvant, such as Freund's adjuvant.
Inoculate at a dose of llllg per CI. The final injection is done by intravenous route 3 days before removing the tile cells for hybridization.

Other steps: fusion with myeloma cells, detection of hybridomas secreting antibodies directed against the immunizing antigen, storage of these hybridomas, cloning and production of antibodies are carried out according to known methods.

Furthermore, the subject matter of the invention is the AC obtained by the above method.
M, especially murine or human ACM.

It is.

Additionally, these monoclonal antibodies may be used for diagnostic purposes to detect, identify or administer Gram(-) bacteria, endotoxins of Gram(-) bacteria or the lipid A-core portion of endotoxins in Gram(-) bacteria. For this reason, the present invention further relates as its subject to the use of said antibodies as well as diagnostic compositions for the detection, identification or administration of said antigens.

Furthermore, these monoclonal antibodies are
It is also used for the purification of products containing non-specific antigenic parts of bacteria. The invention therefore further provides as its subject the use of said AcM for purification, as well as compositions intended for the purification of products with non-specific antigenic parts of Gram(-) bacteria, which are said to be It is characterized by the use of monoclonal antibodies.

Cell hybridoma lines according to 6D5.12BB and DOB4 were purchased from the Collection National de Culture de Microorganismes (CNC) in Barrie.
M-Passeur Institute) on November 5, 1986, with accession numbers 1618, ■619 and I6.
I received 20.

Hybridoma strains registered as 87B3 (anti-E. coli, which also reacts with Serratia and Pseudomonas aeruginosa), F22B6 (anti-Proteus) and H7A2 (anti-Enterobacter) are also available at The Collection in Barrie.
National de Calculebuchera Nilamonia CI
The strain P 52145 was developed by the applicant on June 1981.
It has been deposited with the CNCM under accession number J-163 on May 25th.

The strain of E. coli 0111B4 is well known, especially its mutant strain J5, and has been deposited with different microorganisms by various companies.

[Example] The present invention will be described below with reference to Examples, but is not limited thereto.

Example 1 ■ Preparation of antigen (1) Preparation of biomass Nitriptone broth cultured in a vat at 15 in the following medium: Soybean casein Casein tributin hydrolyzate 17g #Soybean papain decomposition peptone 3g/l chloride sodium
5 (J/oligopotassium phosphate
2.5g/e dextrose
2.5 g/e This culture was carried out by adjusting the I)H to approximately 7.3 after inoculation with the lyophilized strain stored on agar medium and resuspending in physiological fluid.

(2) Collection of bacterial cells After culturing for 15 hours, the bacterial cells were centrifuged for 30 minutes at 500 g, washed twice with a non-pyrogenic solution of 9% NaC; It was dissolved in water 11.

(3) ■ Lotus Next, the bacterial cells were heated to 70°C for 1.5 hours. After cooling to room temperature, 1.25 () of ethylenediaminetetraacetic acid, 1 g of sodium mercury thionate, and 8 d of egg white norizozyme (100 (1/j)) were added to perform bacteriolysis.

This solution was allowed to stand at room temperature for 8 days and then freeze-dried.

(4) Preparation of antigen Next, add 5e acetone to the bacterial powder, and 1.
Stirred for 5 hours. After decanting, the powder was filtered through a type 62 fritted glass and dried under reduced pressure at 40° C. for 16 hours.

The powder was then treated with methanol at 5 for 1.5 hours under stirring. The residue was filtered and dried as before.

The cells treated in this way were then dissolved in water (200#) and kept at 4° C. under stirring for 16 hours.

The suspension was then centrifuged at 11.500 g for 45 min and the supernatant was filtered through Hello Fiber Amicon HtP.
The product was purified by ultrafiltration using a 1OO type membrane.

The antigen is constituted by the fraction retained by these membranes, which is washed with 40 volumes of exchanged water and 0.2
It was filtered through two breast membranes.

After completion of this ultrafiltration, the solution was diluted with 11,500 g of
Centrifuged for minutes and then lyophilized.

This procedure allows the generation of antigens starting from the following strains (see Table 1): Nibrotius vulgaris strain CI P 54160
Proteus mirabilis strain CIPAZ34 Proteus morganii strain CIPA231 Enterobacter cloacae strain CI P 6085 Enterobacter aerogenes strain CI P 6086 Enterobacter aerogenes strain CI P 6023 Etschierkya coli 0111 B4 strain CI P 521 68 Klebsiella nilamonia CI P 52145 strain For example, the antigen was further treated as shown in Examples 1 and 2 of EP 49182 (treatment with cetyltrimethylannimoum bromide, centrifugation, ethanol dialysis, lyophilization and gel filtration) or Processed as in Example 1 of patent no. 0115988.

■ Antibiotic isolated from Klebsiella nilamonia by parenteral route on day 2 and day 21, French patent No. 2,
BAI B/C mice (8 weeks old) were immunized with 20 mcg of the antigen described in No. 540,136; and then with 10 mcg of the same antigen on day 35.

Three days after this last injection, one bladder was removed.

(2) Preparation of tile cells The above lungs were ground with a polygrinder, and the cell suspension was adjusted to 10×1O' cells per ml in Durbecco's medium.

IV. Generation of Myeloma SP210-Ag14 Cultures of 5P20 cells in the proliferative phase were centrifuged, washed, and then lysed in Durbecco's medium at a rate of 8 x 106 cells/d.

(2) Female 15 x 106 SP20 cells and 50 x 106 murine tile cells were centrifuged for 5 minutes at i. toorpm. Remove the supernatant and add 45% polyethylene glycol
1000 (Merck 9729) was added dropwise to the cell residue. This operation was carried out at room temperature and lasted exactly 1 minute.

During the next minute, the residue was broken up with a pipette tip. Then 2rn1 of Durbecco's medium was added dropwise at a rate of 1d per minute, followed by 18d containing fetal bovine serum (10% final).
Durbetzko medium of 4.5mf per minute! was added at a rate of

The cell suspension was homogenized with a pipette and placed in a Petri dish with a diameter of 8 cm. These cells were exposed to 8% C
Cultures were incubated at 37°C in a humidified incubator containing O2. After 18 hours of culture, the suspension was cultured at 2.5 x 105 cells/in Durbec's medium containing 10% fetal bovine serum.
The cell suspension was adjusted to 100 mL per well, and the cell suspension was placed in a plate with 24 wells (Costar) at a volume of 0, ttml per well.
distributed in the proportion of After 6 hours of incubation at 37°C in a humidity-saturated atmosphere containing 8% CO2, 8 wells were cultured for 1 d.
Durbecco (Gibco 0411965) supplemented with selective medium of
9 (7! Fetal live serum ioy
Penicillin 100U/m! Streptomycin 100mcM glutamine
2mH pyruvate
1mM Hyboxanthin 5xlO-
5M Azaserine 10-58 These cells were cultured under the same conditions as above.

0.2 d of fresh selection medium was added every week. After approximately 2 weeks when colonies could be identified with the naked eye, the culture supernatant was removed from the 8 wells.

Vl, Detection of Secreting Hybridomas Hybridomas secreting antibodies directed against the substance that served as the last iteration to immunize the mice were detected by subjecting these supernatants to the F1iSa test for this antigen. Carbonate/bicarbonate buffer (pH 9,6-0,05M
Imuron microelisa plate (Dynachiku 92430:
I put it in the 8 hole of Marnes U Coquette. After 18 hours of incubation at 4° C. in a humid chamber, the contents from the wells were removed and washed twice with the same buffer solution.

300 μC of a 0.1% solution of live serum albumin (BSA Sigma Chemical Company, St. Louis, Mo.) in carbonate buffer was deposited. After 2 hours of incubation at 37°C in a humidified chamber, the liquid was removed and
and PBS buffer containing 0.5% Tween 20 (
Washing was performed three times with pl-17,4-0,01M).

The supernatant of the hybridoma to be tested was diluted with PBS Tween, added at a rate of 20 μe per well, and incubated at +4°C.
The cells were cultured for 18 hours.

After four washes with PBS Tween, the alkaline phosphatase-marked antibody murine anti-immunoglobulin was removed.
．． Diluted in PBS Tween containing 1% bovine serum albumin and then added at a rate of 200 μe per well.

After culturing for 2 hours at room temperature and then washing 4 times, paranitrophenyl phosphate (Sigma) 20011e
(jethanolamine buffer 1 mg/m, 119.8)
added. The reaction between an enzyme and its substrate is carried out for 30 minutes at room temperature.
Continued for 1 minute and then stopped by adding 3M sodium hydroxide solution of 5011e. A positive reaction is indicated by yellow coloration and its optical density can be determined at 405 nm.

Vl, 10% of hybridomas containing the desired antibody in the stored supernatant of secretory hybridomas
Durbetzko's medium containing fetal bovine serum was added first to 26-well plates and then to Petri dishes.

When the number of cells was sufficient, the cell suspension was adjusted to approximately 4 x 10 B cells per d of fetal bovine serum containing 10% dimethyl sulfoxide (DMSO). The hybrid thus prepared was stored in liquid nitrogen.

(2) Cloning The stored hybridomas were then cloned by limiting dilution to specifically select each clone secreting the antibody. At this stage, antibody secretion was measured in the culture supernatant by the E1iSa test described in Vl above. The obtained clones were stored in liquid nitrogen.

ix, Production of antibodies (a) Production in mice 6xlO-5 secretory cells were transferred to the BAL via the intraperitoneal route.
Injected into a B/C rat, this rat received 0.5 15 days ago.
ml of bristane (2,6,10,14-te(~ramethylpentadecane)) was administered. Ascites was removed 10-15 days after injection of the cloned cells and then incubated at 200 O
Centrifuged at rpm.

The ascites fluid collected in this way was directly subjected to Elisa analysis (
(see Section V1 above) and characterized the therapeutic activity of these antibodies in conditions of endotoxic shock in mice (see Section V1).

(b) Production in vitro This production was also carried out in vitro in small bottles or vats.

The isotypes of monoclonal antibodies present in the culture supernatant were identified by IgM, IQA, IQGt, IgG2a, I
Immunization of murine immunoglobulins directed against gG2b, IgG3 was carried out by the double diffusion method on agar (Ouchtelony) with light or heavy anti-linkage (see Table 2).

(b) Antigen specificity.
No. 907, No. 2,462,477, No. 2,490,4
The lipopolysaccharide of Klebsiella nilamonia and the bacterial species described in No. 96 and No. 2.574.429@ were tested.

The results are shown in Table 3 (Example 1).

Xl, Isolation of Monoclonal Antibodies and 1(a) Antibodies present in the supernatants of cultures carried out in vitro or in ascites fluid were precipitated with 45% ammonium sulfate and then dissolved in Tris buffer (HCN 50m) I) I let it happen. Then,
This solution was first equilibrated with DEAE in the same buffer.
Chromatographed on a Sephadex column. The fractions not retained on this column contain immunoglobulin purines of isotype G.

(b) Purification of antibodies of various G isotypes could be carried out by chromatography on Sepharose Protein A or Ultragel Protein A.

In this method, the supernatant of a cell culture or ascites fluid is
Added directly to the column containing the immunoadsorbent in phosphate buffer.

IgG remained fixed to protein A, whereas type M and A immunoglobulins were not retained. The selected eluates IgG1, IgG2a, IgG2b, IgG3 were purified with buffer as described in the technique of Ei IP, L, Ei et al. (1978), Immunochemistry, Vol. 15, pp. 429-436]. 1q by continuously washing the column.

The properties of immunoglobulins purified in this way (specificity,
isotype, endogenous toxin shock-neutralizing activity) No. Vl,
Analyzed as described in sections X and X■.

Xl, Preparation of immunoadsorbents and their use Purified antibodies were immobilized on rods of Sepharose (Pharmacia) or Ultraglue (IBF), which had been previously activated with cyanogun bromide or epoxy. French Patent No. 2,490 , No. 496 and No. 2.574,429
The antigens or fragments thereof described in this issue were purified by affinity chromatography on solid supports thus prepared.

Xl, Therapeutic Uses of These Antibodies The model chosen to demonstrate this effect is described by Galanos [Proceedings of the National Academy of Sciences LJSA, Vol. 76, No. 11 (1
979), pp. 5939-5943].

B6 D2 F1 mice were inoculated by the intravenous route with 0.2 d of antibody-containing ascites fluid and then 3 hours later by the intravenous route with Klebsiella nilamonia endotoxin mixed with 8 mg of galactoseamine. In parallel, a group of comparison animals was inoculated with the same volume of ascites fluid from mice inoculated with cells SP210-Ag14 instead of the antibody-containing ascites fluid.

The results are expressed as the number of surviving animals [see Table 3 (Example 1)].

Table ■ Example 1: Protection in endogenous toxic shock Monoclonal antibodies against Klebsiella nilamonia Number of surviving mice Isocell Type Specificity Treatment group Comparison group 6D5 1
C] G3 10/10 10/1012Be
IgGA 10/10 0/10 Example 2
: Animals were immunized with E. coli antigen.

■ Preparation of antigen (see Example 1) ■ Immunization with Escherichia coli antigen BALB/C mice ( 8 mice) were immunized. 3
On day 5, injections of purified LPS-LAP antigen were given at a rate of 20 mcg per mouse. 3 of this iteration
A day later, the lungs were removed.

(2) Preparation of tile cells The lungs were ground with a polygrinder, and the cell suspension was adjusted to 10×10 6 cells per 1 d in Durbetzko medium.

IV, i Myeloma SP21O-Aq14 (7) Cultures of 5P20 cells in the proliferative phase were centrifuged, washed, and then lysed in Durbecco's medium at a rate of 8 x 10 cells/d.

(2) Furrow 15×10B SP20 cells and 50×106 Nesmi tile cells were centrifuged at 1,1100 rpm for 5 minutes. The supernatant was removed and 1 d of 45% polyethylene glycol 1000 (Merck 9729) was added dropwise to the cell residue. This operation was carried out at room temperature and lasted exactly 1 minute.

During the next minute, the residue was broken up with a pipette tip. Then 2d Durbetzko medium at 17 per minute! and then 18 m containing fetal bovine serum (10% final).
1 of Durbecko's medium was added at a rate of 4.5 d/min.

Homogenize the cell suspension with a pipette and
It was placed in a Petri dish of m. These cells were incubated at 8% CO2
The cells were cultured at 37°C in a humid incubator containing After 18 hours of culture, the suspension was reduced to 2.5 x 10 cells/rd in Durbecco's medium containing 10% fetal bovine serum. and the cell suspension was distributed into plates with 24 wells (Costar) at a ratio of 0.47 per well. After 6 hours of incubation at 37°C in a humidity-saturated atmosphere containing 8% CO2, a selection of 1d Durbecco (Gibco 0411965) with the following composition in each formula:
9 (7! Fetal bovine serum 107 Penicillin 100U/d Streptomycin 100mcMml Glutamine
2mH pyruvate
1mM Hypoxanthine 5X1 Kano 5H Azaserine 1 Kano 5H4 These cells were cultured under the same conditions as above. 0.2 h of fresh selection medium was added every week. After about 2 weeks when colonies were visible to the naked eye, the culture supernatant was removed from the 8 wells.

Vl, Detection of Secreting Hybridomas Biblidomas secreting antibodies directed against the substance used in the last iteration of immunizing mice were detected by subjecting the supernatant to an Elisa test against the antigen. Carbonate/bicarbonate buffer (pH 9,6-0,05M> Solution of Nickel Antigen 250/Zj on Imron's MicroELISA plate with 96 holes (Dynate 92430: Marnes U.) After 18 hours of incubation at 4° C. in a humidified chamber, the contents of the wells were removed and washed twice with the same buffer solution.

Approximately 300 μe of a 0.1% solution of live serum albumin (BSA Sigma Chemical Company, St. Louis, Mo.) in carbonate buffer was added. After 2 hours of incubation at 37°C in a humid chamber, the liquid was removed and
and PBS buffer containing 0.5% Tween 20 (
Washing was performed three times with II 7.4-0.01 M>.

The supernatant of the hybridoma to be tested was diluted with PBS Tween and added at a rate of 200 μC per well and incubated for 18 hours at +4°C.

After four washes with PBS Tween, alkaline phosphatase-marked antibody murine anti-immunoglobulin was diluted in PBS Tween containing 0.1% bovine serum albumin and then added at a rate of 200 μe per well. Ta.

After incubating for 2 hours at room temperature and then washing 4 times,
μe paranitrophenyl phosphate (Sigma),
(lmMd in jetanolamine buffer, pH 9,8>
added. The reaction between an enzyme and its substrate is carried out for 30 minutes at room temperature.
It lasted for a minute and then was stopped by the addition of 50μe of 3M sodium hydroxide solution. A positive reaction is indicated by a yellow coloration and the optical density can be determined at 405 nm.

Vl, the stored supernatant of the secreting hybridoma contains 10% of the hybridoma containing the desired antibody.
cultured in Durbecco's medium containing fetal bovine serum,
They were first added to a 24-well plate and then to a Petri dish.

When the number of cells was sufficient, the cell suspension was adjusted to approximately 4 x 10 cells per d of fetal bovine serum containing 10% dimethyl sulfoxide (DMSO). Saved with.

(2) Cloning The stored hybridomas were then cloned by limiting dilution to specifically select each clone secreting the antibody. At this stage, antibody secretion was measured in the culture supernatant by the EIiSa test described in Vl above. The clones thus obtained were stored in liquid nitrogen.

IX, Production of antibodies (a) Production in mice 6 x 10 secretory cells were transferred to BALB by intraperitoneal route
/C note the mouse, this mouse grew 0.5m 15 days ago.
fl of pristane (2,6,10,14-tetramethylpentadecane) was inoculated. 10-15 days after injecting the cloned cells, the ascites was removed and then 2000 r
Centrifuged at pm.

The ascites fluid collected in this way was directly subjected to Elisa analysis (
(see Section V1 above) and characterized the therapeutic activity of these antibodies in conditions of endogenous toxic shock in mice (see Section V1).

(b) Production in vitro This production was also carried out in vitro in small bottles or vats.

X. Characterization This specificity has been demonstrated to be useful for E. coli of various different serotypes (iso), Serratia marsetuscens (Sigma Klebsiella nilamonia (List), Klebsiella nilamonia 01, K2 (C1052145), Nilamonia aeruginosa). 06 (CI P59.39), 011 (
CIP59.44), 012 (CIP59.45
> LPS [0, Zeitschrift Nazir Holschung (1952) by Westphal, Vol. 7b, pp. 148-155]. Further tests were conducted for comparison.

The results in Table 1V show that the antibodies of the species (04B, D
5 B5, D5 D2, DOB3, D9 A2
) discriminates LPS of different serotypes of E. coli and other antibodies (De B4 , D11A5 , D7 B3 )
It has also been shown that LPS of other Enterobacteria such as Celadea or Klebsiella can be identified (Table IV A). Antibody 87 B3 is serotype 06
．． 011.012 Pseudomonas aeruginosa L
It reacts strongly against PS and all bacterial cells of the same serotype, and also against serotype M (P, aeruginosa ATCC 21636).
) also reacts (1st VB term).

Xl, Isolation and Purification of Monoclonal Antibodies (a) Antibodies present in supernatants of in vitro cultures or ascites fluid were precipitated with 45% ammonium sulfate and then dissolved in Tris buffer (HCN, 50 mM). . This solution was then chromatographed on a DEAF Sephadex column previously equilibrated with the same buffer. The fractions not retained on the column contain immunoglobulins of isotype G.

(b) Purification of antibodies of various G isotypes can be carried out by chromatography on Sepharose Protein A or Ultraglu Protein A.

In this method, cell culture supernatant or ascites fluid was added directly to the column containing the immunoadsorbent in phosphate buffer.

IQG remains anchored to protein A, whereas type M and A immunoglobulins are not retained.

Its selective elution IgG1, IgG2a, IgG2), I
FG3 as a technique of stingray [P, L., et al. (1978), Immunochemistry, Vol. 15, pp. 429-436]
was obtained by successive washings of the column with a buffer similar to that described in .

The properties of the thus purified immunoglobulin (specificity, isotype, endogenous toxic shock neutralizing activity) were analyzed as described in Sections VLX and X■ above.

X■, Preparation of immunoadsorbent and its use The purified antibody was immobilized on a rod of Sepharose (Pharmacia) or Ultragel (IBF) which had been activated in advance with cyanobromide or epoxy. French patent. Nos. 2,490,496 and 2,574,4
The antigens or fragments thereof described in Publication No. 29 were purified by affinity chromatography on the solid support thus prepared.

xm, Therapeutic Uses of These Antibodies The model selected to demonstrate this effect is the Galanos [Proceeding National Academy of Sciences U.
SA, Volume 76, No. 11 (1979), No. 5939~
Page 5943].

0.2 ml by intravenous route for B6 D2 F1 rats.
antibody-containing ascites fluid, followed 3 hours later by intravenous route with Klebsiella nilamonia endotoxin mixed with 8 mg of galactoseamine. In parallel, a group of comparison animals was inoculated with the same volume of ascites fluid from mice inoculated with cells 5P210-AC]14 instead of the antibody-containing ascites fluid.

The results are expressed as the number of surviving animals.
I C] G3 10/10 1/10 Cells De B4 secrete immunoglobulins that protect against endogenous toxic shock.

Example 3: Animals were immunized with Proteus or Enterobacter antigens (Table 1).

(See Examples 1 and 2 for each item: ■ ■ ■ V ■ I I ■ X I X■).

X. Characterization The specificity of the antibodies was tested against antigens prepared from Proteus or Enterobacter strains. Antibody E1
1B5 discriminates antigens made from three Proteus strains (Table V) and antibody H7A2 discriminates antigens made from three Enterobacter strains (see Table V1).

■ Anti-E. coli monoclonal antibody protection against experimental infection.

1. Materials and Methods Ten fiOF1 mice (IFF, A-
ORFDO) group received 0.9% NaC 90 minutes before injection.
50% silica (Kieserguhr Merck, code 8119) M in l2 was injected intraperitoneally.

Two E. coli strains, namely C)I'll:B4(
C, I, p, 52167) and 02:KI:H
4 (C, I, P, 54.52) in tryptone-casein-soy broth (Merieux Laboratories) at 37 °C.
Cultured for 8 hours. These were then diluted with 0.9% NaCN to the required concentration and 0.5 ml of this suspension was injected intraperitoneally. The exact number of bacteria was tested by plate counting.

2, E. coli 0111:B4, significantly protected mice were administered two antibodies D683 and D6B4 3 hours before injection (Table VI). The effective dose was estimated by administering antibody De B4 at a dose range of 10...cg to 1...0 per 41 screws. Protection was obtained even at doses as low as 50 mcg per 41 screws (Part Ⅰ).
table).

Injection of the antibody De B4 1 hour after injection gave significant protection, although it was less effective than in the prophylactic treatment (Table 1X).

In case of heterogeneous infection with E. coli 02, D
A clear increase in mean survival time was observed for animals inoculated with OB3 antibody.

E. coli 0111:84 in a group of 10 Swiss rats.
Different amounts of ammonium sulfate precipitated antibodies were injected intravenously 3 hours before infection.

The method, comparison and results are shown in Table VI.

■, anti-P, monoclonal antibody Protection against experimental infection.

Ten 1ltlOF1 mice aged 5-6 weeks (IFFA
-CREDO) group was used. K. Nilamonia 01:2 was cultured on nutrient agar for 18 h at 37°C, then the bacterial suspension was diluted to the desired concentration with 0.9% NaC,
And 0.5 d of this suspension was intraperitoneally injected into a rat.

Treatment with monoclonal antibodies.

Monoclonal antibodies were purified by two rounds of ammonium sulfate precipitation to 45% saturation. PB without pyrogenic substances
After dialysis with S, the concentration of monoclonal antibodies was determined by the radiation immunodiffusion technique described by Mancini.

Claims (13)

[Claims] (1) In preparing mammalian monoclonal antibodies directed against the lipid A-core portion of endotoxins of various types of Gram(-) bacteria, the antigen that acts on immunization of immune-competing cells is lipid A- Gram with the core part cut out (
-) A method for producing a monoclonal antibody characterized in that it is a complete bacterial lipopolysaccharide. (2) The method according to claim 1, characterized in that the complete lipopolysaccharide is obtained from bacteria of the species Escherchia, Klebsiella, Pseudomonas, Enterobacter, Bacteroides, Serratia or Proteus. (3) The method according to claim 2, characterized in that the complete lipopolysaccharide is obtained from Klebsiella pneumonia. (4) The method according to claim 2, characterized in that the complete lipopolysaccharide is obtained from E. coli. (5) The method according to claim 1, 2, 3 or 4, characterized in that the lipopolysaccharide is present as a lipopolysaccharide protein structure in combination with lipid A. (6) A monoclonal antibody obtained by the method according to any one of claims 1 to 5. (7) The monoclonal antibody according to claim 6 obtained from a mouse. (8) The human monoclonal antibody according to claim 6. (9) A hybridoma obtained when carrying out the method according to any one of claims 1 to 5. (10) D_6B_4, 6D_5, 12B_6, B_7
A hybridoma producing a monoclonal antibody selected from the group consisting of B_3, E_2_2B_6 and H_7A_2. (11) A drug comprising the monoclonal antibody according to any one of claims 6 to 8. (12) A pharmaceutical composition or diagnostic composition containing the monoclonal antibody according to any one of claims 6 to 8. (13) For purifying a product containing a non-specific antigenic portion of Gram (-) bacteria, characterized by using the monoclonal antibody according to any one of claims 6 to 8. Composition of.